Accommodation in anuran Amphibia and its role in depth vision

J Comp Physiol A (1986) 158:133-143 Journal of

Sensory, Comparative ~176

Physiology A ~"~176176 physiology

�9 Springer-Verlag 1986

Accommodation in anuran Amphibia and its role in depth vision

R.H. Douglas 1'3, T.S. Collett 1, and H.-J. Wagner 2 1 M.R.C. Group in Neurophysiology, School of Biological Sciences, University of Sussex, Falmer, Brighton, Sussex,

BN1 9QG, United Kingdom 2 Institut fiir Anatomie und Zellbiologie, Universit/it Marburg, Robert-Koch-Stral3e 6, D-3550 Marburg,

Federal Republic of Germany a Present address: Department of Optometry and Visual Science, The City University, Dame Alice Owen Building,

311-321 Goswell Rd, London ECI 7DD, United Kingdom

Accepted October 15, 1985

Summary. 1. In order to analyse the mechanism of accommodation in anurans, drugs (miotic or atropine) were applied to the cornea of anaesthetized animals to change the refractive state of their eyes. During such changes, the lens and cornea were photographed and the refractive state of the eye was measured using laser speckle refractometry. Measurements taken from the photographs confirmed suggestions by Beer (1898) that accommodation is achieved by moving the lens and not by changing the shape of the lens or cornea. The change in refractive state induced by pharmacolog- ical manipulation was about 10 diopters with an accompanying shift in lens position of about 150 gm. Calculations based on a schematic eye suggest a disparity between the amount of lens movement theoretically needed to produce a 10 D shift in refractive state and the amount actually observed.

2. The lens is probably moved by two protractor lentis muscles which are positioned so as to pull the lens towards the cornea (Tretjakoff 1906, 1913). Dissection and HRP preparations revealed that these muscles are innervated by fibres of the oculomotor nerve which relay in the ciliary ganglion. In R. esculenta and R. pipiens, the ciliary ganglion consists of only 8 to 12 nerve cells.

3. MS222 anaesthesia and lymphatic injection of curare cause the lens to move away from the cornea, presumably because they destroy the resting tonus of the protractor lentis muscles. We discuss this finding in relation to the frog's 'resting' accommodative state, and conclude that unpara- lysed frogs are likely to be myopic, and not emmetropic as previous work suggests.

4. Prey capture was analysed in R. pipiens after the disruption of accommodation by bilateral section of the oculomotor nerve. Estimates of prey distance remained accurate when vision was binoc-

ular. However, during monocular vision, when the oculomotor nerve was sectioned on one side and the other eye was either occluded or had its optic nerve cut, frogs consistently underestimated the distance of their prey. This result suggests, in agreement with earlier evidence, that accommodation is used for judging depth when vision is limited to one eye, but that binocular information predom- inates when it is available.

5. Atropine applied to the cornea of monocular frogs also causes distance to be underestimated. It is argued from this that frogs assess distance by monitoring the motor commands sent to their accommodative muscles, rather than by using sensory information from the muscles themselves.

Introduction

Anurans can judge the distance of their prey with precision using monocular as well as binocular cues (Ingle 1972; Podufal 1971; Collett 1977). There is Strong evidence that to assess distance with one eye toads (and presumably frogs) monitor the accommodative state of their eyes when the prey is in focus (Podufal 1971 ; Collett 1977; Jor- dan et al. 1980). Monocular frogs can measure distances from about 5 cm to 20 cm (Collett and Harkness 1982), which implies that they should be able to accommodate over at least 15 diopters. However, there are no measurements of accommodative range in anurans and indeed little is known about the way in which they accommodate. This is surprising, given their pivotal position in the transition from aquatic to terrestrial optics.

In the late nineteenth and early years of this century work by Beer (1898), Hess (1911) and Tret-

134 R.H. Douglas et al. : Accommodation in anuran Amphibia and its role in depth vision

jakoff (1906, 1913) suggested that, as in fish, the eyes of Anura accommodate to objects at different distances by moving the lens forwards or back- wards. Beer stimulated electrically the surface of the cornea of enucleated eyes in the region of the ciliary body. He found that in toads, but curiously not in frogs, the lens shifted forwards. Subse- quently, Tretjakoff described two 'protractor lentis' muscles within the eyes of both frogs and toads which seemed likely to control the position of the lens. These muscles run, one ventrally and one dor- sally, from the corneoscleral junction to the ciliary body. As they shorten they will pull the ciliary body towards the cornea and with it presumably the lens. This idea received support from Hess, who observed that electrical stimulation of the cornea induced two slight depressions at the corneoscleral junction where the protractor lentis muscles insert. He suggested that this buckling of the cornea, which occurred in both frogs and toads, was caused by the contraction of these muscles.

In this paper, we start with a brief description of the protractor lentis muscles and their innervation. Our major concern, however, is to confirm that accommodation is indeed present in anurans and that it is mediated by lens movement. We also provide some estimate of the amount of movement possible. To do this we applied drugs (atropine and miotic) to the cornea so as to relax or excite the protractor lentis and then measured any conse- quent shift in lens position and change in refractive state. This gives a rough indication of the range over which the animal might normally be expected to accommodate. In addition, we describe the effect of MS222 anaesthesia and curare on lens position and discuss this in relation to the animals' 'resting' refractive state. Finally, to show that anurans do use the depth information potentially available in accommodation, we examined prey capture both after the nerves innervating the protractor lentis were severed and after application of atropine to the cornea.

Methods Intraocular muscles and their innervation

Observations were made on adult specimens of Rana esculenta, Rana pipiens and Bufo marinus. The tissue was fixed by perfus- ing anaesthetized animals through the conus arteriosus with a mixture of 2% paraformaldehyde and 2% glutaraldehyde in 0.06 mol/1 phosphate buffer (pH 7.4). In order to locate the ciliary ganglion and the nerves associated with it, the course of the oculomotor (III cranial) nerve was followed under a dissecting microscope from the floor of the mesencephalon to its entry into the orbit. Subsequently, the walls of the orbit were opened and the third nerve dissected out from the sur- rounding extraocular muscles to expose the nerve and its major branches within the orbit.

The accommodative muscles and their innervation were also studied in thick plastic sections. After fixation, heads were bisected sagittally and the entire orbit, including the medial and caudal walls, osmicated (1% for 10 h), dehydrated and embedded in Epon. After warming the cutting face of the block with a tacking iron, 3(~50 ~tm sagittal or horizontal sections were cut on a sliding microtome equipped with a steel blade. Sections were stained in dilute Richardson's solution (Richard- son et al. 1960), rinsed briefly, dried, and mounted on slides.

In order to trace the efferent neural fibres between the intraocular muscles and the ciliary ganglion, 5 Ixl 30% HRP (Sigma, Type VI) was injected into the anterior chamber of both eyes. 24 to 36 h later, animals were anaesthetized and perfused with a mixture of 1% paraformaldehyde and 1% glutaraldehyde. The orbits were then opened ventrally and the head immersed in diaminobenzidine (0.05%) and H202 (0.06%) for 30 to 40 rain. The reaction product was observed directly under the stereomicroseope or in thick plastic sections, prepared as above with osmication omitted.

Effect of drugs on lens position and refractive state of the eye

Three basic experiments were performed: 1. To assess the effect of the MS222 anaesthesia on the

position of the lens, four adult Rana temporaria were immersed in 0.25% MS222 for 10 to 20 min. In order to determine the separation between lens and cornea both eyes were photographed from above every 10 min as the animals started to recover from the anaesthetic.

2. The effect of curare on the position of the lens was examined in four adult Rana pipiens. Animals were anaesthetized in MS222 and the distance between the lens and cornea determined in both eyes at intervals for 60 rain. 0.8 mg of curare was then injected lymphatically and the lens position was monitored for a further 40 min.

3. Adult Rana temporaria, Rana eseulenta and Bufo viridis were anaesthetized by immersion in MS222. The refractive state of the left eye was then determined using laser speckle refractometry and both eyes were photographed from above. After these initial readings, several drops of either atropine (600 tag/ml atropine sulphate) or a miotic (0.01% carbachol and 0.03% neostigmine methyl sulphate in Rana temporaria and Bufo viridis and 1/10th this strength in Rana eseulenta) were applied to both corneas and readministered every 10 rain throughout an experiment. Lens position and refractive state were recorded at regular intervals to assess the action of the drugs.

a Refractive measurements. Most methods for measuring the refractive state of an eye, such as conventional retinoscopy, become difficult when the pupil is small and so cannot be used in conjunction with a miotic which causes severe pupillary con- striction. On the other hand, laser speckle refractometry, the method used here, is sometimes even easier to use with small pupils. The procedure is described in detail by Manteufel et al. (1977) and is only outlined here.

When coherent light from a laser hits an uneven surface, such as the retina, the reflected light produces striking interference patterns of dark and light spots known as laser speckle. Laser speckle refractometry exploits the fact that the appear- ance of the speckle varies characteristically with the size of the area that is illuminated. A large area has a multitude of small surface facets orientated in different directions, thus giving much scope for interferenee and producing an interference pattern of numerous small spots. When the area is smaller, however, the possibilities for interference are reduced and consequently the pattern is composed of fewer but larger spots. Parallel rays of light entering an emmetropic eye are focussed

R.H. Douglas et al. : A~ommodat ion in anuran Amphibia and its role in depth vision

He-Ne laser a

135

~e-Ne l a s e r ~ ' : =* b

Fig. 2a, b. Optical system used in laser speckle refractometry. L1-L4: positive lenses. L3 can be shifted along the optic axis of the system by means of a micromanipulator. M microscope for viewing speckle, bs beam splitter, a The laser light entering the eye is parallel and is focussed on the retina as in an emmetropic eye. b In a hyperopic eye parallel light is focussed behind the retina (dotted line). The beam can, however, be focussed on the retina (solid line) by moving L3

Fig. 1 a, b. Speckle caused by reflection of coherent light from a frog's retina viewed through the microscope of the optical system shown in Fig. 2 focussed in the plane of the pupil, a beam focussed on the retina resulting in large patches of speckle. b beam focussed behind the retina causing the speckles to be small

as a small spot on the retina. The size of the dark and light patches comprising the speckle is then maximum (Fig. I a). However, if the eye is myopic or hyperopic, the image plane of the impinging parallel light is in front of or behind the retina. The spot of light on the retina is thus larger and consequently the speckle size is smaller (Fig. 1 b).

For refractometry, the optical system through which the laser beam passed before entering the eye was adjusted so as to maximize the speckle size. The amount of adjustment needed provided a measure of the degree of ammetropia. The optical arrangement used is shown in Fig. 2a. Light from a Model :156 Spectra-Physics He-Ne 1.0 mW laser (wavelength= 632.8 nm) was collimated by a series of lenses (LI-L4). After leaving L4, the beam passed through a beam splitter and en- tered the eye approximately along its optic axis (50 ~ from the midline, Schneider 1954). The speckle pattern reflected from the eye was viewed through a microscope focussed in the plane of the pupil. Light leaving L4 will be parallel, provided that the distance between L3 and L4 is the sum of the focal lengths of these lenses ( 2 f ; f = 3 cm for both lenses).

If the eye is emmetropic, such parallel light will cause maxi- mal speckle size (Fig. I a) as it is well focussed on the retina. However, if the eye is anametropic, the light will not be focussed on the retina and the speckles will be smaller (Fig. 1 b). The laser light can, however, be brought into focus on the retina in such an ammetropic eye by altering the distance between

L3 and L4. Figure 2b shows this for a hyperopic eye. The more the eye deviates from emmetropia, the greater the change in distance between L3 and L4 necessary to bring the light into focus. The separation between L3 and L4, specifies the degree of ammetropia and the exact refractive state of the eye was calculated using the expression:

D = -- (c-- (f(x - J ) / ( x - 23')) ) -1

(after Manteufel et al. 1977)

where D is measured in diopters, e is the distance between L4 and the cornea, x is the separation between L3 and L4 a n d f i s the focal length of the lenses.

b Measurement of separation between cornea and lens. The distance between cornea and lens was measured from enlarged photographs of the eye. Anurans are conveniently 'pop-eyed', so that the cornea and the anterior surface of the lens are clearly visible when the eye is submerged.

Animals were placed in a Perspex holder to keep them in a constant orientation relative to the camera and briefly submerged in water. The eyes were then photographed from above through a dissecting microscope. Photographic negatives were later projected on to a screen, magnifying the eye approximately 300 fold, so that the distance between the middle of the cornea and the front surface of the lens could be measured accurately (Fig. 7). We looked for changes in the curvature of the cornea and the anterior surface of the lens by superimpos- ing tracings of the eye.

Prey capture after H and III nerve section

Adult Rana pipiens were anaesthetized by immersion in MS 222. The oculomotor nerve was exposed through an inci- sion in the roof of the mouth and sectioned close to where it emerges from the cranial cavity. In three animals the opera- tion was performed bilaterally. In another three animals, the oculomotor nerve was sectioned unilaterally, and the optic nerve cut on the other side.

One or two days later, prey capture could again be elicited. Animals in a mat black arena were recorded on video tape, using a camera suspended above the arena, while they attempted to catch meal worms or dummy prey which were presented

136 R.H. Douglas et al, : Accommodation in anuran Amphibia and its role in depth vision

on the ground at various distances in front of them. Frogs with bilateral section of the oculomotor nerve were first tested with binocular vision. Subsequently, one eye was occluded by a piece of aluminium foil and prey presented to the other eye alone. The foil was later removed and the animal retested bin- ocularly. Finally, the optic nerve on the other side was sectioned to test the previously occluded eye. This whole procedure took about four weeks. Two measurements were obtained from the record of each attempted prey capture: 1. The distance between the prey and the animal's eyes the frame before the animal began its lunge towards the prey (target distance). 2. The distance between the eyes before any movement occurred and the position of the tongue at its furthest extent during prey capture (snapping distance).

In an additional experiment, the accuracy of depth judge- ments was examined in a monocular Rana pipiens with and without atropine applied to the cornea.

Results

The protractor lentis muscles and their efferent innervation

Both dorsal and ventra l p r o t r a c t o r lentis muscles are cylindrical, a b o u t 300 Ixm long and 60 g m in d iameter when fixed. They were c o m p o s e d of lon- gitudinally a r r anged s m o o t h muscle cells sur- rounded by a thin layer o f connect ive tissue and are loosely embedded in the t rabecula r m e s h w o r k tha t fills the i r idiocorneal angle (Fig. 3). One end of the muscle at taches at the corneoscleral junc t ion wi thout dis turbing the a r r angemen t of the collagen fibres in the corneal s t roma. The muscle te rminates in a foo t plate which seems to be fas tened to the endothe l ium or to the equivalent o f Descemet ' s m e m b r a n e . At the other end, the muscle cells in- termingle with f ibroblas ts and merge with the con- nective tissue o f the ciliary body , oppos i te to the site where the zonula fibres p r o b a b l y emerge.

In all ver tebra tes studied so far, the innerva t ion of the a c c o m m o d a t i v e muscu la tu re is by way o f fibres f r o m the third cranial nerve which relay in the ciliary gangl ion before penet ra t ing the sclera. We therefore tried to locate this p a t h w a y in Anura . Gross dissection revealed tha t in Rana esculenta and Rana pipiens the third nerve enters the orbi t t h rough the medial wall near the orbi ta l f loor and a b o u t 1 m m f r o m the caudal wall. Soon af ter it enters the orbi t the nerve gives of f two m o t o r branches. Just distal to this poin t it bulges, fo rming a swelling f rom which several fine nerves emerge. These run towards the eye in close, p rox imi ty to the opt ic nerve and per fora te the sclera at several points on the pos te r ior surface o f the globe.

In thick plast ic sections, accumula t ions o f 8-12 neurons were observed associated with the oculo- m o t o r nerve at a locat ion which cor responds well with the swelling no ted above dur ing gross dissec-

Fig. 3. Thin plastic section (1 gm) showing the dorsal protractor lentis muscle of the frog, R. pipiens. * indicates a nerve in cross section at the base of the iris. The lens would be attached to the ciliary processes which are shown in the center of the bottom edge of the figure

Fig. 4. Thick plastic section (40 gm) of the medial and posterior region of the orbital cavity near the entry of the oculomotor nerve in R. eseulenta. Halfway between the optic nerve and the origin of the lateral rectus muscle a cluster of four perikarya can be seen which are part of the ciliary ganglion. Thin bundles of osmicated nerve fibres are associated with this ganglion, a Muscle origins, b Branch of oculomotor nerve, c Ciliary ganglion. d Myelinated nerve fibres, e Lateral rectus muscle

t ion (Fig. 4). We feel tha t this s t ructure is analo- gous to the ciliary gangl ion o f higher vertebrates , a c la im s t rengthened by observa t ions on the H R P injected prepara t ions . Per ikarya within this g roup of cells conta in react ion p roduc t when H R P is injected into the anter ior chambe r of the eye: the H R P taken up by m o t o r nerve fibres te rminat ing on the in t raocular muscles is t r anspor ted re t rogra- dely a long axons to the cell bodies in this ciliary ganglion.

Effect of drugs on lens position and refractive state

In order for the image of objects viewed at var ious distances to be focussed on the retina, animals can

R.H. Douglas et al. : Accommodation in anuran Amphibia and its role in depth vision 137

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Fig. 5a, b. Changes in lens/cornea separation of the left and right eyes during recovery from MS222 anaesthesia, a R. temporaria; b R. pipiens injected with 0.8 mg curare 60 min (*) after anaesthesia was induced

either change the radius of curvature of the refractive surfaces (lens and cornea) or alter the distance between these surfaces and the photosensitive layer. We show here that a muscle stimulant (miotic) and a relaxant (atropine) applied to the cornea of frogs and toads change the distance between the lens and retina, but have no effect on the radius of curvature of either the anterior surface of the lens or the cornea. Consistent changes in refractive state were associated with the effects of these drugs on lens position. When the lens moved towards the cornea, the eye became accommodated for close objects (myopic shift in refractive state), whereas with tens movement away from the cornea, the eye became accommodated to more distant objects (hyperopic shift). The effects de- tailed here are most likely caused by the action of atropine and miotic on the neuromuscular junc- tions of the protractor lentis muscles.

1. MS222/Curare

During recovery from anaesthesia the distance between lens and cornea gradually shortened as both R. pipiens and R. temporaria passed from deep to light anaesthesia (Fig. 5). This result suggests that anaesthesia causes the protractor muscles to relax, so that the lens shifts away from the cornea, and that during recovery tonic activity is restored to the muscles. Curare has a similar effect. When in-

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jected during recovery from MS222 anaesthesia the separation between lens and cornea increased (Fig. 5). Any drugs applied to an anaesthetized or paralysed animal are thus probably acting on muscles which are already either completely or partially relaxed.


Fig. 7. An eye of R. esculenta photographed from directly above the animal before (left) and 30 min after (right) the application of a miotic 1 -cornea, 2-anlerior chamber, 3-lens protruding through the iris, 4-iris

There are marked species differences in the action of MS222. The difference between the resting refractive state in an awake animal and one under MS222 anaesthesia is much greater in R. temporaria than it is in either R. esculenta or B. viridis (Table 2), suggesting that MS222 relaxes the protractor lentis more completely in R. temporaria than it does in the other species. As we show below, this variation is reflected in the action of atropine. Interestingly, anaesthetics vary in their relaxing effect. Animals anaesthetized with ether were much less hyperopic (Table 2).

2. Atropine/miotic

R. temporaria. Lens position and refractive state were both unaltered by the application of atropine (Fig. 6a). Any effect this drug might have seems to be masked by the powerful action of MS222 in relaxing accommodation. Miotic, however, caused the lens to move towards the cornea with a corresponding myopic shift in refractive state (Figs. 6a, 7).

Bufo viridis. Application of atropine was followed consistently by an increase in the distance between.

lens and cornea, together with a hyperopic shift in refractive state (Fig. 6b). However, a transient movement of the lens towards the cornea, with an accompanying myopic shift, always preceded the more prolonged and pronounced movement of the lens away from the cornea. Miotic, on the other hand, induced an immediate myopic shift and movement of the lens towards the cornea (Fig. 6b).

Rana esculenta. Atropine increased the distance between lens and cornea and caused a hyperopic shift in refractive state (Fig. 6c). In the long term, miotic decreased the separation between lens and cornea. However, in 7 out of 9 animals, the first effect of miotic was to increase slightly the separation between lens and cornea (mean 26.5 gin, +_ 12.7). This initial response of the lens was not accompanied by a hyperopic shift; the first change of refractive state in response to miotic was consistently in a myopic direction. These drug-induced changes in lens position and refractive state are summarised in Table 1. Despite some minor differences, the major effects of atropine and the miotic were clear-cut and similar in all species. Miotic


T a b l e 1. Effect of miotic and atropine on the cornea/lens separation and refractive state of the eye in 3 species of anuran Amphi- bia

Species

R. temporaria B. viridis R. esculenta

Atropine

lens shift (gin)

refractive state (D)

Miotic

lens shift (gm)

refractive state (D)

Total

lens shift (lam) refractive

state (D)

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caused the lens to move towards the cornea and a myopic shift in refractive state, while atropine resulted in the lens moving towards the retina and a hyperopic shift in refractive state�9 Together the drugs induced a maximum lens movement of about 150 gm with a shift in refractive state of about 10 diopters�9 Superimposed tracings of the eye before and after application of the drugs, on the other hand, revealed no change in the curvature of either the anterior lens surface or the cornea.

Effect of H and III nerve section and atropine on prey capture

When both binocular and monocular frogs snap at prey on the ground they usually hit the prey accurately with their tongue (e.g. Fig. 9). Binocular frogs with both oculomotor nerves cut were almost equally accurate (Fig. 8 a). However, when one eye was occluded, these same frogs dramatically snapped short (Fig. 8a). Accurate prey capture was restored once the eye-cover was removed�9 Sim- ilarly, frogs with the optic nerve cut on one side and the oculomotor nerve sectioned on the other also snapped consistently short of their prey.

The anatomical evidence suggests that oculo-

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Fig. 8a, b. Relation between target distance and snapping distance in two R. pipens after IIIrd nerve section. Each data point represents a single attempt to catch prey. a Frog with bilateral IIIrd nerve section. Top: binocular vision. Middle: right eye sees. Bottom: left eye sees. b Monocular frog with unilateral IIIrd nerve section on seeing side. a and b each show data from just one animal, however, the other four animals behaved like one of these two examples.

motor nerve section should destroy the frog's ability to accommodate. Thus the data in Fig. 8 support earlier evidence that accommodation is used to assess the distance of prey when vision is monocular, but that other depth cues are available when vision is binocular.

Nonetheless, the behavioural consequences of oculomotor nerve section were not completely clear-cut. In some animals the relationship between


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Fig. 9. Prey capture in a monocular R. pipiens with and without corneal application of atropine

target and snapping distance was essentially ' f lat ' (Fig. 8 b). Frogs behaved as though the prey were at the same short distance whatever the actual target distance. Interestingly, these animals also snapped at large objects such as a human fist. Other animals, however, while they still snapped short, did show some increase in snapping distance as target distance grew (Fig. 8 a). There are at least two possible explanations for this effect. The first is that the oculomotor nerve is not the only nerve supplying input to the ciliary ganglion and that in some operations this other input was also severed. The second is that other, weaker, monocular cues supplement accommodation and can only be detected when accommodation is disrupted.

Figure 9 shows the relation between target distance and snapping distance with and without the application of atropine. Atropine, which disrupts accommodation (see above), clearly causes the frog to undershoot its target. With atropine the slope of the regression line relating snapping distance to target distance is 0.533, without atropine it is 0.772. This difference is highly significant ( P < 0.001, t =4.277, d.f. = 136) and suggests once more that the accommodative system supplies depth information.

Discussion

ls changing lens position the only mechanism for adjusting refractive state ?

Our experiments have shown that the anuran lens moves through about 150 ~tm but does not change shape. Is this movement of the lens sufficient to account for the 10 D refractive change we have observed?

Calculations based on a schematic eye for Rana esculenta (du Pont and de Groot 1976) indicate that a one diopter change in refractive state of the eye theoretically requires a 27.8 ~tm shift in lens position. In fact, we find that on average a one diopter change in the same species is accompanied by a much smaller shift in lens position (mean: 12.6 ~tm, SD: +5.4 ~tm, n:7). If we accept the accuracy of both our measurements and of the schematic eye, it would seem that in our experiments movement of the lens is not the only factor controlling refractive state. The partial dissociation between lens motion and refractive state observed in R. esculenta treated with miotic argues the same way.

One possibility, for which there is some evidence, is that drugs cause a change in the axial length of the eye, i.e. the separation between cornea and retina. Du Pont and de Groot (1976) noted that when frogs were given Flaxedil the separation between cornea and retina decreased by 90 txm. Flaxedil cannot act directly on smooth intraocular muscles since it blocks the action of acetylcholine at nicotinic receptors, which are only found on striated muscle (Burn 1975). It would therefore have to act either directly on the extraocular muscles or indirectly on the smooth intraocular muscles via the nicotinic synapses of the ciliary ganglion. Although the protractor lentis muscles could not change the axial length of the eye, smooth muscle cells reported to be present in the choroid of Anura (Walls 1942), might influence the position of the retina (Sivak 1980). However, it is improba- ble that such alterations in the axial length of the eye are a normal feature of anuran accommodation. Thus, 10 diopters may be an overestimate of the frog's normal accommodative range.

What is the refractive state of the "resting' eye ?

Anurans, like many of us, spend much of their time doing nothing. What refractive state should an eye adopt during such periods of inactivity? It is useful to know the resting refractive state because it is likely to be the 'starting point ' of any accommodative change.

R.H. Douglas et al. : Accommodation in anuran Amphibia and its role in depth vision

Table 2, Average ' resting' refractive states of 4 anuran species under various conditions

141

Species

R. temporaria B. viridis R. esculenta B. marinus

After MS222 immersion using + 12.67 + 5.06 + 8.83 laser refractometry (7, +2.97) (9, +0.99) (9, -+2.45)

After ether anaesthetizing using + 4.06 - - laser refractometry (3, _+ 3.68)

In an awake animal using + 5.17 +4.67 +4.0 conventional retinoscopy (3, +0.58) (6, -+0.68) (6, +0.32)

Approximate orbital 5 5 7 diameter (ram)

+ 1.25 (4, _+0.29)

/1

All these figures are averages. First value in brackets shows number of eyes exanained, the second figure is the standard deviation

An answer to this question is possible if we assume that the visual system should be ready to detect objects over as wide a range of distances as possible. The argument hinges on a quantity known as the hyperfocal distance. Consider the image on the retina of an emmetropic eye, which does not accommodate to an object approaching it from infinity. As the object moves closer, the image will be focussed further and further behind the photosensitive layer, giving an increasingly blurred image on the retina. But the blurring of the image is not detected until the object is brought within the hyperfocal distance (H). As far as the visual system is concerned, all objects from infinity to H are seen equally clearly without any need for accommodation. One factor which determines H is the limited resolution of the photoreceptors.

H also depends on those geometrical properties of the eye which determine the size of the blur circle. These are the focal length of the eye (J) and the diameter of the aperture (a). It can be shown (e.g. Rolls 1968) that H=f*f/nc, where c is the diameter of the allowable blur circle and n is the relative aperture (f/a). This expression tells us that H will drop sharply with the size of the eye, so that small animals with smaller eyes will have greater depth of focus than larger ones (Green et al. 1980; Collett and Harkness 1982). For the frog, R. pipiens, H is about 20 cm.

If the lens is set to focus on objects placed at the hyperfocaI distance, then objects will be in focus from infinity to half the hyperfocal distance. The far point of the system is then at infinity, the near point at HI2. Thus, an eye can survey the greatest possible range of distances when it is focussed on objects at the hyperfocal distance. This argument leads one to expect that all resting eyes will be myopic, with small ones more so than large.

The greater depth of focus afforded by small eyes means that small animals, probably more inter- ested in seeing close objects, will automatically be equipped to do so without sacrificing the ability to detect distant predators.

At first sight the available evidence contradicts this suggestion. Retinoscopy indicates that anurans, like almost all vertebrate species examined to date, are hyperopic (Hughes 1977). The values obtained for Anura range with the species and author from + 9 . 0 D to - 1 . 0 D (Hirschberg 1882; Beer 1898; Millodot 1971, 1974; Krueger and Moser 1971; Moser and Krueger 1972; du Pont and de Groot 1976; Manteuffel et al. 1977). Our values, obtained using conventional retinoscopy performed on unanaesthetized animals (Table 2), are similar. However, retinoscopy probably does not give a true representation of the refractive state of an eye. Glickstein and Mitlodot (1970) first noted that the smaller the eye, the more retinoscopic readings depart from emmetropia. They suggested that an error arises because the reflective layer is not at the receptors, but somewhat closer to the cornea, perhaps at the interface between retina and vitreous humour. The present results also show a relation between eye size and hyperopia. The species with the largest eye, B. marinus, was more nearly emmetropic than the two other species with smaller eyes (Table 2). Although this general relationship has now been shown to be true for many different species, the precise location of the reflecting layer is still uncertain (Hughes 1977).

Our measurements of the 'resting' refractive state obtained using laser speckle refractometry also overestimate the degree of hyperopia since: 1. Readings were taken from anaesthetized animals with reduced muscle tonus. 2. As with conventional retinoscopy, the identity of the reflecting layer


is unknown. 3. Because of the chromatic aberration of the frog's lens (Millodot and Sivak 1978), the long wavelength of the laser will exaggerate still further the apparent hyperopia.

The current consensus is that far from being hyperopic most vertebrates are emmetropic. This view is based largely on the fact that R. pipiens and R. temporaria, even though they appear hyperopic when tested retinoscopically, are seen to be emmetropic when examined by methods which do not rely on reflected light (Millodot 1971, 1974; Moser and Krueger 1972). Unfortunately, these experiments were performed on animals immobilized with curare, which we have shown relaxes the accommodative muscles (Fig. 5) as it does in fish and mammals (Meyer and Schwassmann 1970; Westheimer and Blair 1973). Du Pont and de Groot (1976), contrary to Krueger and Moser (1971), also saw a relaxing effect of Flaxedil. They found that R. esculenta immobilized with Flaxedil were retinoscopically 5 D more hyperopic than freely moving individuals. Thus the emmetropia observed by Millodot (1971, 1974) and Moser and Krueger (1972) in paralysed individuals, with relaxed accommodative muscles, implies that awake frogs are actually myopic, as one might reasonably expect for animals with such small eyes.

How is accommodative state monitored?

The present results agree with earlier evidence that accommodative cues provide estimates of distance when vision is monocular. They also allow us to say something about the way in which the accommodative state is monitored to provide this information. Animals could in principle do this either by sensing the position of the lens or by monitoring the motor commands to the protractor lentis. The frog's estimates of prey distance after atropine application help distinguish between these alterna- tives.

With the protractor lentis muscles partially relaxed by atropine, more powerful motor signals than usual will be needed for the muscles to gener- ate a given amount of force. The motor signals which will then bring a target into focus would, under normal conditions, be those appropriate for a somewhat closer target. Thus, if a frog were to use the motor command to the protractor lentis as an index of distance, atropine should lead the frog to snap short of its prey.

If the animal were to monitor lens position, we would expect one of two outcomes. Suppose atropine does not prevent the frog from focussing on a target, but merely requires the production

of unusually powerful command signals. Lens position and sensory feedback would then be normal, as should the frog's estimate of distance. On the other hand, should the muscle be so relaxed that the frog cannot contract it sufficiently to focus on close targets, the lens would then be in a position appropriate for more distant objects and the frog should 'overshoot' its target. There is one major uncertainty in this argument: we do not know whether atropine might also influence this hypo- thetical sensory input.

Figure 9 shows that after the application of atropine frogs clearly undershoot their prey. The linear relationship between the snapping and target distances, which exists despite the presence of atropine, suggests that the frog continues to focus on its target, but has to exert more effort than usual to do so. We conclude, with the important caveat mentioned above, that anurans gauge their accommodative state by monitoring motor outflow rather than sensory inflow.

Jordan et al. (1980) also found that atropine causes toads to undershoot their prey. They inter- preted this result rather differently and suggested that the toad approaches a moving prey until it is so close that the eye can no longer keep the image of the prey in focus. At this moment the toad snaps. But, as Fig. 9 suggests, the information available in the anuran's accommodative state can in fact be used to provide a real cue to distance, allowing animals to judge how far away their prey lies and to organize the appropriate behaviour to reach it.

Acknowledgements. We are grateful to W. Himstedt, G. Man- teuffel and C. Werner for advice on the use of laser speckle refractometry, D. Finch for help with photography, T. Griffiths for assisting with refracting and G.L. Ruskell for helpful discus- sions. R.H.D. and T.S.C. were supported by a grant from the S.E.R.C.

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Accommodation in anuran Amphibia and its role in depth vision

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